David biweekly meeting
project updates
Project 4: Reconstitution of endocytic actin network
recent progress
EM
- currently processing hopefully final set of samples for EM
- I'm optimistic that I solved the issue of beads moving around during EM
processing by:
- incubating fixed samples on dish overnight instead of 1 hour
- stringently washing away loosely attached beads in buffer 3 times before light micrsocopy
- marking regions where beads do not move upon shaking the stage back and forth during light microscopy imaging
- due to optimus prime bright field not working anymore, I worked out a new
imaging scheme on the airyscan
- pro: whole-slide tile scans work really well
- con: need to manually register fluorescence and bright field channels, though they get distorted slightly differently from each other so it can't be a perfect overlay
- example tile scan of +Las17 dish
bright field
cyan lipid, magenta Sac6-GFP
time lapse fluorescence imaging
- purpose: to help interpret the deformed membranes I've observed by EM in the +Las17 +LatA condition
- finished collecting +/- LatA time lapses on airyscan
- 3 biological replicates
- 1-2 movies per replicate, ~1 hour each
- ~10 beads per movie
- worked out strategy for systematically 3D visualizing and reproducibly
recording vesiculation events on each bead
- I do an unbiased screen for all movies by hiding the Sac6 channel and only looking for membrane budding events in the lipid channel
- so far, analyzed 10-20 beads in each condition and only observed obvious membrane budding in +Las17 -LatA condition
- however, there are stable membrane tubules/irregularities in all conditions
- example vesiculation event in +Las17 -LatA condition
- example membrane irregularity in +Las17 +LatA condition
next steps
- continue EM processing and imaging
- continue scoring fluorescence time lapse data
Project 7: Mechanical regulation of CME by protein LLPS
feedback from lab meeting?